SUMMARY OF WORK This new research area involves a model of in vitro differentiation of cardiomyocytes originating from embryonic stem cells (R1) and embryonic carcinoma cells (P19). The research is aimed at understanding the structure-function relationships of the sarcoplasmic reticulum calcium release channel and its relationship to spark formation and calcium movements in developing myocardial cells. We have been able to successfully differentiate pluripotent ES and EC cells into embryoid bodies containing contracting cardiac-like cells. Application of retinoid acid to the differentiating cells has been reported to result in a population that is predominantly ventricular in nature, and this pharmacological intervention is being exploited to study calcium movements in early ventricular like cells. To identify, atrial versus ventricular like cells, expression vector constructs are being constructed that link ventricular markers to the green florescent protein (GFP). These constructs will be introduced into the cells and positive transformants identified through neomycin resistance selection. This work is being performed in collaboration with the Excitation Contraction Coupling Unit of the Laboratory of Cardiovascular Science. Their contribution has involved the techniques of voltage clamping and confocal microscopy to analyze calcium spark formation, calcium transients and function of calcium handling proteins.